Skip to main page content
U.S. flag

An official website of the United States government

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2013 Feb;24(3):285-96.
doi: 10.1091/mbc.E12-06-0441. Epub 2012 Dec 5.

A dominant mutation in mec-7/β-tubulin affects axon development and regeneration in Caenorhabditis elegans neurons

Leonie Kirszenblat  1 , Brent Neumann, Sean Coakley, Massimo A Hilliard
Affiliations

A dominant mutation in mec-7/β-tubulin affects axon development and regeneration in Caenorhabditis elegans neurons

Leonie Kirszenblat et al. Mol Biol Cell. 2013 Feb.

Abstract

Microtubules have been known for decades to be basic elements of the cytoskeleton. They form long, dynamic, rope-like structures within the cell that are essential for mitosis, maintenance of cell shape, and intracellular transport. More recently, in vitro studies have implicated microtubules as signaling molecules that, through changes in their stability, have the potential to trigger growth of axons and dendrites in developing neurons. In this study, we show that specific mutations in the Caenorhabditis elegans mec-7/β-tubulin gene cause ectopic axon formation in mechanosensory neurons in vivo. In mec-7 mutants, the ALM mechanosensory neuron forms a long ectopic neurite that extends posteriorly, a phenotype that can be mimicked in wild-type worms with a microtubule-stabilizing drug (paclitaxel), and suppressed by mutations in unc-33/CRMP2 and the kinesin-related gene, vab-8. Our results also reveal that these ectopic neurites contain RAB-3, a marker for presynaptic loci, suggesting that they have axon-like properties. Interestingly, in contrast with the excessive axonal growth observed during development, mec-7 mutants are inhibited in axonal regrowth and remodeling following axonal injury. Together our results suggest that MEC-7/β-tubulin integrity is necessary for the correct number of neurites a neuron generates in vivo and for the capacity of an axon to regenerate.

PubMed Disclaimer

Figures

FIGURE 1:
FIGURE 1:
Posterior neurite outgrowth defects in ALM neurons of ky852 mutants. Morphology of ALM in wild type (A), ky852 heterozygotes (B), and ky852 homozygotes (C). White arrowheads indicate the ectopic posterior process. (D) Quantification of ALM phenotypes, classified as normal (no posterior process), moderate (medium length posterior process), and severe (long posterior process). Each data set is based on an n of at least 100 animals. Scale bar: 25 μm.
FIGURE 2:
FIGURE 2:
Ectopic neurites in ky852 mutants develop following embryogenesis. Morphology of ALM neurons in wild type and ky852 mutants in the embryo (A and B), at hatching (C and D), and 5 h after hatching (E and F). (G) Quantification of ALM neurons with posterior neurite defects in ky852 mutants during developmental stages corresponding to the images in (A–F). Each data set is based on an n of at least 100 animals. Error bars represent the SE of proportion. Scale bars: 20 μm.
FIGURE 3:
FIGURE 3:
The ky852 phenotype is caused by a mutation in the mec-7 gene. (A) Diagram of the mec-7 gene. Solid bars represent exons and connecting lines represent introns. The ky852 mutation and two other alleles that cause similar ALM neurite outgrowth phenotypes are indicated. (B) Rescue of ALM defects by insertion of wild-type mec-7 into mec-7(ky852) mutants. Two independent lines that were generated with different concentrations of injected transgene are shown. Phenotypic distributions between transgenics and nontransgenic controls were significantly different in both cases (chi-square test: p < 0.0001). (C) Induction of mec-7(ky852) phenotype by insertion of mutant mec-7 into wild-type animals. Two independent lines that were generated with different concentrations of injected transgene are shown. In both cases, transgenics had a reduced percentage of normal ALMs and an increased percentage of ALMs with intermediate length posterior process defects (Fisher’s exact test: p < 0.0001). (D) ALM phenotype of mec-7(u278) homozygotes, mec-7(u278) heterozygotes, and mec-7(u278)/mec-7(ky852) double heterozygotes. Data sets for (B–D) are based on an n of at least 100 animals.
FIGURE 4:
FIGURE 4:
Manipulation of microtubule stability using colchicine and paclitaxel affects development of ectopic ALM neurites. (A) Rescue of ALM posterior neurite defects in mec-7(ky852) animals grown on colchicine throughout development. The distribution of ALM phenotypes in mec-7(ky852) animals grown on colchicine was significantly different from that of control mec-7(ky852) animals (chi-square test: p < 0.0001). (B) Rescue of ALM defects in mec-7(ky852) animals when treated with colchicine during the first 24 h but not the second 24 h after hatching. Animals treated with colchicine from 0 to 24 h were significantly different than controls (chi-square test: p < 0.0001). (C) Ectopic neurites generated in ALM neurons of wild-type (bus-17; zdIs5) animals grown on paclitaxel. ALM phenotypes of animals treated with paclitaxel were significantly different than controls (Fisher’s exact test: p < 0.03). Each data set is based on an n of at least 50 animals in (A) and (B) and 59 and 24 animals for paclitaxel-treated and control animals, respectively, in (C).
FIGURE 5:
FIGURE 5:
Genetic interactions of mec-7(ky852) with MAPs and Wnts. (A) ALM phenotypes in double mutants of the microtubule-binding proteins, PTL-1/Tau and UNC-33/CRMP2, and the kinesin-like molecule VAB-8. mec-7(ky852); unc-33(e204) and mec-7(ky852); vab-8(e1017) double mutants have significantly more wild-type ALMs and less severe ALM defects compared with the mec-7(ky852) mutant (chi-square test: p < 0.001). (B) ALM phenotypes in mec-7(ky852); cwn-1; cwn-2 triple mutants. The mec-7(ky852) mutation converts the reversed unipolar neurons generated in cwn-1; cwn-2 double mutant animals into bipolar neurons. Each data set is based on an n of at least 100 animals.
FIGURE 6:
FIGURE 6:
Distribution of RAB-3 and UNC-104/kinesin in ALM neurons of mec-7(ky852) mutants. (A) Normal localization of the RAB-3::mCherry presynaptic marker in ALM neurons of wild-type animals. White arrowheads indicate RAB-3 in the nerve ring branch of ALM. Gray dashed circles represent coelomocytes expressing GFP that were used as a transgenic marker. (B) RAB-3::mCherry localizes to the posterior processes of ALM neurons in mec-7(ky852) mutants. White arrowheads indicate RAB-3 in the nerve ring branch and ectopic posterior process of ALM. (C) UNC-104::GFP localization in ALM neurons of wild-type animals. Localization of UNC-104 is indicated (white arrowheads). (D) UNC-104::GFP localizes to the posterior processes of ALM neurons in mec-7(ky852) mutants. (E) Quantification of RAB-3::mCherry localization on the anterior processes and on the posterior processes of ALM neurons. (F) Quantification of UNC-104::GFP localization on the anterior and posterior processes of ALM neurons. Each data set is based on an n of at least 50 animals. Scale bar: 25 μm.
FIGURE 7:
FIGURE 7:
Axonal regeneration defects in mec-7(ky852) mutants. Regeneration of the anterior ALM process in wild-type animals and both the anterior and posterior ALM processes in mutants is shown in each graph. (A) Percentage of ALM neurons showing regrowth following axotomy in wild-type and mec-7(ky852) animals. (B) Quantification of the average length of regrowth of regenerating axons shown in (A). (C and D) Percentage of ALM neurons showing reconnection of the regenerating process to the distal fragment, and of these reconnected axons, the percentage showing maintenance of the connection (fusion). (E and F) Images showing axonal regeneration in a wild-type animal (E) and defective axonal regeneration a mec-7(ky852) mutant (F). In wild-type animals, the regenerating axon regrows from the proximal fragment (closest to the cell body) to the distal fragment of the ALM neuron. n for each data set is indicated on the bars of the graph. Error bars represent the SE of proportion. *, significant difference between wild-type and mutant animals (Student’s t test: p < 0.05).
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Abe N, Cavalli V. Nerve injury signaling. Curr Opin Neurobiol. 2008;18:276–283. - PMC - PubMed
    1. Adler CE, Fetter RD, Bargmann CI. UNC-6/Netrin induces neuronal asymmetry and defines the site of axon formation. Nat Neurosci. 2006;9:511–518. - PMC - PubMed
    1. Baas PW. Neuronal polarity: microtubules strike back. Nat Cell Biol. 2002;4:E194–E195. - PubMed
    1. Baas PW, Lin S. Hooks and comets: the story of microtubule polarity orientation in the neuron. Dev Neurobiol. 2011;71:403–418. - PMC - PubMed
    1. Baran R, Castelblanco L, Tang G, Shapiro I, Goncharov A, Jin Y. Motor neuron synapse and axon defects in a C. elegans alpha-tubulin mutant. PLoS One. 2010;5:e9655. - PMC - PubMed

Publication types

MeSH terms

LinkOut - more resources